In studying physiology of microorganisms it is important to vary only the parameter the effects of which are being studied. For example, if the effect of temperature change is being studied then pH and other parameters should be fixed and remain constant. In addition, microorganisms under the study should not be affected by stress caused due to the lack or excess of the substrate. If the transfer from one reactor to another is slow, an uncontrolled growth of microorganisms will take place in transfer hoses—the residual substrate will be consumed, the starvation will follow which in turn will lead to activation of various stress responses (microbial culture is no longer in a state of stable physiology). If in the beginning of the experiment microbial culture is in unstable physiological state there is always a possibility that the result is not due to the variable parameter. Thus, the constancy of physiological state after the culture transfer is an important criterion in all bioreactors within the system if the wish is to carry out experiments or production processes in identical starting conditions. Similarly, the physiological state should remain constant in the phase of increasing the volume to obtain unambiguous response to changes in environmental parameters throughout later cultivation. If in case of continuous cultivation a microbial culture is in unstable physiological state at the starting point of the experiment a stabilisation phase, which usually is five residence times, must precede. Thus, if the physiological state is altered accidentally during cultivation process (increasing of the volume or transfer of microbial cultures) duration of the experiment will increase by a stabilisation phase.
Several cultivation technologies are known using different bioreactor systems. Continuous, batch and fed batch cultivations have been used the most in interconnected bioreactors.
A system consisting of one 5 L mother reactor and of six 1 L daughter reactor(s) was described in the article “A multi-bioreactor system for optimal production of malaria vaccines with Pichia pastoris” J. Fricke, K. Pohlmann, F. Tatge, R. Lang, B. Faber, R. Luttmann, Biotechnology Journal 2011, vol 6 (4), pp 437-451. An increase of the volume of biomass in fed batch cultivation takes place in the mother reactor, after that microbial culture is transferred to the daughter reactor. The disadvantage of this solution is alteration of other parameters besides volume in the mother reactor (micro-organisms are processed with methanol to induce synthesis of recombinant protein). Also, during increase of the volume the temperature in mother reactor is lowered from 30° C. to 20° C. resulting in unstable physiology of microorganisms. The transfer is followed by regrowing of the microbial population volume in the mother reactor, but due to the effects of previous methanol and temperature alterations new microbial culture has potentially different physiological state compared to the original culture. In addition, this solution does not enable the preservation of the same physiological state in mother- and daughter-reactors after the transfer. One solution described in the article prescribes a 30° C. temperature in mother reactor and 20° C. in daughter-reactor, which consequently leads to different physiological state. Stabilisation to the new conditions can be observed in the results of the study, as the concentration of methanol fluctuated after the transfer of culture. The second solution prescribed an identical initial temperature in mother- and daughter reactor(s), but in daughter reactor the microbial culture was diluted twofold with a fresh feed. Although the transfer was conducted in a periodical manner, different environmental conditions were applied in some daughter-reactors (temperature, pH, concentration of methanol). Thus, environmental parameters are abruptly changed during the transfer, which results in stress response of microorganisms. Detailed description of the technical solution is not presented in this article, so it can be assumed that the rapid transfer rate of microbial culture was not considered a critical parameter. However, it is important to keep in mind that during the fed batch cultivation, where concentration of biomass is high and consequently so is the oxygen consumption rate, an anaerobic environment is very likely to occur in case of a slow transfer which in turn results in changes in physiology. Possible problems in maintaining aerobic environment can be seen from a fluctuating QO2 line in FIG. 7 of the article.
In the article “A Two-stage CSTR Cascade for Studying the Effect of Inhibitory and Toxic Substances in Bioprocesses”, R. Hortsch, C. Loser, T. Bley, “Engineering in Life Sciences”, 2008, vol 8 (6), pp 650-657 a cultivation system with continuous transfer between reactors is described. This solution does not allow a periodical transfer of microbial culture. Since the flow of biomass in daughter reactor is twice as high as in the mother reactor due to additional substrate necessary for growth of the biomass, cultivation method and consequently the physiological state of microorganisms is not the same in mother and daughter reactor(s) after transfer. A slow peristaltic pump was used for the biomass transfer in this solution, which is not fast enough to transfer biomass with unchanging physiology.
The article “Continuous, high-level production and excertion of a plasmid-encoded protein by Escherichia-coli in a two-stage chemostat”, J. Fu, D. B. Wilson, M. L. Shuler, “Biotechnology and Bioengineering”, 1993, vol 41 (10), pp 937-946 describes a system with continuous transfer of microbial culture from mother- to daughter reactor, with different dilution rates in two reactors and a continuous induction of protein synthesis with IPTG (Isopropyl β-D-1-thiogalactopyranoside) in daughter reactor. Due to differences in environmental conditions in mother- and daughter reactor(s) it is not possible to perform a transfer of microbial culture without changing its physiological state, resulting in different conditions in mother- and daughter-reactors from the beginning of the experiment.
Article “Bioreactor mixing efficiency modulates the activity of a prpoS::GFP reporter gene in E. coli”, F. Delvigne, M. Boxus, S. Ingels, P. Thonart, “Microbial Cell Factories”, 2009, vol 8 describes a system with a fed batch cultivation and continuous transfer of biomass from stirred tank reactor to plug flow reactor to imitate heterogeneity of the content of industrial bioreactor (different gradients of nutrients or other environmental parameters in different parts of bioreactor). Concentrated glucose solution was added to microbial culture in plug flow reactor and the culture was not aerated. In this case, a transfer of microbial culture from one reactor to another is continuous, meaning that in daughter reactor the experiment cannot be started with a culture stabilised in the mother reactor. Different cultivating methods are applied in different reactors (chemostat in stirred tank reactor and batch cultivation in plug flow reactor). From the plug flow reactor biomass is directed back to the stirred tank reactor, thus altering a physiological state of the fed batch culture in mother reactor.
In the article “Genome evolution and adaptation in a long-term experiment with Escherichia coli”, J. E. Barrick, D. S. Yu, S. H. H. T. K. Oh, D. Schneider, R. E. Lenski, J. F. Kim, “Nature”, 2009, 461, pp 1243-1247 the most widely used reactor-to-reactor transfer solution is described, where batch cultivation is repeated in order to achieve a greater number of microorganisms generations. In the phase of exponential growth bacteria are planted into a sterile culture medium and nurtured after what transfer and the rest of procedure are repeated. During the transfer a dilution of microbial culture takes place, resulting in stress response of microorganisms. In addition, stirring is suspended in bioreactor during the transfer, which leads to decline of oxygen solubility and along with that to changes in microbial physiology.
Bioreactor systems where transfer of microbial cultures is applied occur in several patent documents. The biggest disadvantage of these solutions so far is that they do not allow the transfer of microbial culture in strictly controlled environment thus in different reactors the physiological state is different. Solutions described in the following patent documents are good examples for that.
German patent application DE19547656A1 describes sequential reactors to detect toxic substances from wastewater. The transfer of microbial culture between reactors is described, but preservation of constant physiological state is missing. The transfer is continuous, physiological state varies in different reactors which means that the experiment cannot be started in daughter reactor with microbial culture stabilised in mother reactor. During cultivation the volume in mother reactor remains constant. This solution does not allow the optimisation of the process of microbial transfer. The UK patent GB1270006 describes the series of reactors where microbial culture is kept in continuous logarithmic growth phase, meaning that due to the effect of diluting with media, concentrations are altered during the transfer and physiological state is not constant. Japanese patent application JP8229534A describes the transfer of culture from one reactor to another focusing not on the fixed physiological state at the beginning of the experiment but rather on obtaining different summary conversion rates in different reactors. Patent application WO2005042694A2 describes a system of transfer of microbial culture between bioreactors where physiological state of microorganisms is altered during the process. Different cultivation methods are applied in different reactors which makes starting the experiment with a stabilised culture impossible. Solution described in Bulgarian patent BG50222A does not allow the transfer of microbial culture in controlled conditions nor preservation of the physiological state. The USA patent application US2010041124A1 describes a multi-stage bioreactor system where microorganisms are transferred between reactors, but since medium is inserted only in the first reactor this solution does not allow to start experiments with the same physiological state in different reactors. Biomass concentration in different reactors is not identical.
Solutions known to date do not allow the transfer of microbial culture between different reactors in controlled conditions nor using the same cultivation method which makes preservation of the physiological state, environmental conditions and biomass parameters during the transfer impossible. Due to different cultivation methods in different reactors the solutions known to date render it impossible to start cultivations after the transfer with a stabilised culture in different reactors. Due to different environmental conditions in different reactors it is impossible to transfer microbial culture without interfering its physiological state and the experiment cannot be started in daughter reactor with conditions identical to those of the stabilised culture in mother reactor.